Currently, it is thought that as the alveoli in the lungs collapse and expand, the SPs are desorbed and adsorbed to the lung surfactant,
16,19 and it is worthwhile considering if our data indicate that a similar process may be occurring in the TFLL. As background, it must be kept in mind that the lipid components of lung surfactant are polar and almost entirely phosphatidylcholine,
17 which contrasts to the composition of meibum, which is primarily waxes and cholesterol esters.
4,5 The consequence of this fundamental difference is that phospholipids tend to form a monolayer on the surface and under high pressure collapse into a bilayer in the form of vesicles that move into the subphase,
43,44 whereas meibum forms a duplex film with molecules that move off the surface going to lenses at the air interface.
45 Our data suggest that the protein molecules are moving from the subphase into the lipid layer and at a minimum this can be interpreted as reducing the area per molecule occupied by the lipids. This puts pressure on the lipids to move off the surface. This is similar in principle to slowly adding lipids to the surface while maintaining a fixed area as was done by MacDonald and Simon
43 using phosphatidylcholine. The transition from monolayer to bilayer begins for phospholipids at π ∼ 15 mN/m, and at approximately 50 mN/m all of the lipids exist as a bilayer.
43,44 This leads to a curve showing the relationship between either the average molecular area per molecule or surface pressure and the ratio between the lipids existing at the surface and those desorbed from the surface as a bilayer (Fig. 3 in Feng et al.
44 ). The monolayer and bilayer lipids are in equal amounts at approximately 29 mN/m.
43,44 Once desorbed in this manner, the phospholipid vesicles do not return to the surface as a monolayer in the timeframe typical of isocycles used here.
38,43 With films of lung surfactant, a similar desorption into the subphase is represented as a flattening of the curve during isotherms
16 and is again modeled as multilayer formation, which allows layers of phospholipids to be pushed into the subphase stabilized by SPs. These layers can return to the surface, but many are released into the subphase as unilamellar vesicles to be recycled in situ by macrophages and alveolar type II cells. Compared with lung surfactant, the SPs interact very differently with meibomian lipids. The constant and increased pressure during isocycles of mixed films of SPs and meibomian lipids indicate that they are not being desorbed and reabsorbed. If this had been the case, a flattening of the curve at smaller surface areas and a π
max similar to that of meibomian lipids alone would have been expected because this would represent desorption. With meibomian lipids, the stability of the isocycles through different temperatures and a similarity in appearance to normal meibomian lipid films suggest that they are acting as true surfactants in the TFLL and remain at the aqueous lipid interface. Alternatively, they may be desorbed into the upper phase of the lipid layer where they would most likely form lenses, but we are unable to determine if this was the case using our techniques. Nevertheless, the presence of SPs would lower the surface tension of the TFLL, even in low concentrations, and if the concentrations of SPs were increased, meibomian lipids appear to be able to tolerate this. However, this view might not reflect what is happening in vivo because of high compression rates and ratios during a blink. It appears that by rapidly compressing a film of bovine lung surfactant extract coating the surface of a captive bubble, the proteins stay in the film and the film does not collapse. Instead it becomes metastable (glass).
46 Similar rapid compression experiments using meibomian lipids might resolve this.